Vaccine composition comprising Flt3-ligand

ABSTRACT

Flt3-ligand can be used to generate large numbers of dendritic cells from hematopoietic progenitor and stem cells. Flt3-ligand can be used to augment immune responses in vivo, and expand dendritic cells ex vivo. Such dendritic cells can then be used to present tumor, viral or other antigens to naive T cells, can be useful as vaccine adjuvants. When flt3-L is used and/or administered in combination with other reactive agents, e.g. CD40 binding proteins, 4-1BBL or antibodies reactive with 4-1BB, CD30 ligand antagonists, RANKL, and/or interferon alpha the combination further enhances immune responses and the effectiveness of vaccine adjuvants.

RELATED APPLICATIONS

This application is a Continuation application of pending priorapplication Ser. No. 10/397,687 (Conf. No. 7724) filed Mar. 25, 2003;which is a Divisional of U.S. application Ser. No. 10/241,927, filedSep. 11, 2002; which is a Continuation of U.S. application Ser. No.09/444,027, filed Nov. 19, 1999 (now abandoned); which is aContinuation-In-Part of U.S. application Ser. No. 09/154,903, filed Sep.17, 1998 (now abandoned); which is a Continuation-In-Part of U.S.application Ser. No. 08/725,540, filed Oct. 3, 1996 (now abandoned);which is a Continuation-In-Part of U.S. application Ser. No. 08/539,142,filed Oct. 4, 1995 (now abandoned); the disclosure of each of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a dendritic cell stimulatory factor, tomethods of enhancing an immune response in vivo, methods of expandingdendritic cells ex vivo, and to preparations of purified dendriticcells, and to dendritic cell populations useful in the manipulation of Tcell-mediated and B-cell mediated immune responses.

BACKGROUND OF THE INVENTION

The objective of vaccination is to provide effective immunity byestablishing adequate levels of antibody and a primed population ofcells that can rapidly expand on renewed contact with antigen. The firstcontact with antigen during vaccination must not be injurious to therecipient and thus usually consists of pathogenically-deficient antigen.

A frequent difficulty with active immunization protocols is that thevaccine antigen does not possess sufficient immunogenicity to promote astrong immune response, and therefore a sufficient level of protectionagainst subsequent challenge by the same antigen. In addition, certainantigens may elicit only weak cell-mediated or antibody responses. Formany antigens, both a strong humoral response and a strong cell-mediatedresponse is desirable.

For decades, researchers have experimented with diverse compounds toincrease the immunogenicity of vaccines. Immunopotentiators, also knownas adjuvants, of vaccines are compositions of matter that facilitate astrong immune response to a vaccine. In addition, the relatively weakimmunogenicity of certain novel recombinant antigens has requiredadjuvants to be more potent. Vaccine adjuvants have different modes ofaction, affecting the immune response both quantitatively andqualitatively. Such modes of action can be by mobilizing T cells, actingas depots and altering lymphocyte circulation so that these cells remainlocalized in draining lymph nodes. They may also serve to focus antigenat the site of immunization, thereby allowing antigen specific T cellsand B cells to interact more efficiently with antigen-presenting cells.They may also stimulate proliferation and differentiation of T cells andhave effects on B cells, such as enhancing the production of differentIg isotypes. Further, adjuvants may stimulate and affect the behavior ofantigen-presenting cells, particularly dendritic cells and macrophages,rendering them more effective for presenting antigen to T cells and Bcells.

Dendritic cells are a rare and heterogeneous cell population withdistinctive morphology and a widespread tissue distribution. Adiscussion of the dendritic cell system and its role in immunogenicityis provided by Steinman, R. M., Annu. Rev. Immunol., 9:271-296 (1991),incorporated herein by reference. Dendritic cells display an unusualcell surface phenotype and can be characterized by the presence of thecell surface markers CD1, CD4, CD86, CD11c, DEC-205, CD40 or HLA-DR, andthe absence of CD14 and other lineage markers. Dendritic cells have ahigh capacity for sensitizing MHC-restricted T cells and provide aneffective pathway for presenting antigens to T cells in situ, bothself-antigens during T cell development and foreign antigens duringimmunity. Thus, there is growing interest in using dendritic cells exvivo as tumor or infectious disease vaccine adjuvants. See, for example,Romani, et al., J. Exp. Med, 180:83 (1994). The use of dendritic cellsas immunostimulatory agents has been limited due to the low frequency ofdendritic cells in peripheral blood, the limited accessibility tolymphoid organs and the dendritic cells' terminal state ofdifferentiation. Dendritic cells originate from CD34+ bone marrowprogenitors, and the proliferation and maturation of dendritic cells canbe enhanced by the cytokines GM-CSF (sargramostim, Leukine®, ImmunexCorporation, Seattle, Washington), TNF-α, c-kit ligand (also known asstem cell factor (SCF), steel factor (SF), or mast cell growth factor(MGF)) and interleukin-4. Therefore, an agent that stimulated thegeneration of large numbers of functionally mature dendritic cells invivo or in vitro would be of wide importance.

SUMMARY OF THE INVENTION

Flt3-ligand (“flt3-ligand,” “flt3-L,” “Flt3-L”) is known to affecthematopoietic stem and progenitor cells. It was surprisingly found thatflt3-ligand can also potently stimulate the generation of downstream orintermediate, cells such as myeloid precursor cells, monocytic cells,macrophages, B cells, and dendritic cells from CD34+ bone marrowprogenitors and stem cells. The present invention pertains to a methodof mobilizing dendritic cells in vivo, expanding dendritic cells ex vivoand to preparations of dendritic cells. The preparation of purifieddendritic cells according to the invention would potentially find use asvaccine adjuvants. Also included within the embodiments of the inventionis a method of preparing antigen-specific T cells using the dendriticcells mobilized with flt3-ligand.

The invention provides for the use of an effective amount of flt3-ligandto increase or mobilize the numbers of intermediate cells in vivo, forexample, in the patient's peripheral blood or spleen. While theinvention relates to the generation of large numbers of such downstreamand intermediate cells (e.g., myeloid cells, monocytic cells andmacrophages) from CD34+ cells using flt3-ligand, the focus isparticularly on dendritic cells. By increasing the quantity of thepatient's dendritic cells, such cells may themselves be used to presentantigen to T cells. For example, the antigen may be one that alreadyexists within the patient, such as a tumor antigen, or a bacterial orviral antigen. Flt3-L may be used, therefore, to increase the numbers ofdendritic cells in vivo to boost a patient's immune response againstexisting antigens. The invention further provides for using combinationtherapy to enhance a patient's immune response. Such combination therapyincludes administering flt3-ligand and one or more therapeutic reagentsin amounts sufficiently effective to upregulate the patient's immuneresponse. Alternatively, flt3-ligand may be administered prior to,concurrently with or subsequent to administration of an antigen to apatient for immunization purposes. Thus, as a vaccine adjuvant,flt3-ligand can generate large quantities of dendritic cells in vivo tomore effectively present the antigen. The overall response is a strongerand improved immune response and more effective immunization to theantigen.

The invention also provides a method of generating large quantities ofdendritic cells ex vivo. Following collection of the patient's CD34+hematopoietic progenitors and stem cells, flt3-ligand can be used toexpand such cells in vitro (also known as ex vivo expansion) and todrive such CD34+ cells to differentiate into dendritic cells of thelymphoid or myeloid lineage. The resulting collection of dendritic cellscan be administered to a patient to provide a stronger and improvedimmune response to an antigen. Alternatively, the resulting dendriticcells find use as a vaccine adjuvant and can be administered prior to,concurrently with or subsequent to antigen administration.

The invention also provides a method of generating large quantities ofantigen-presenting dendritic cells ex vivo. Following collection of thepatient's CD34+ hematopoietic progenitors and stem cells, flt3-ligandcan be used to expand such cells in vitro and to drive such CD34+ cellsto differentiate into dendritic cells. The resulting collection ofdendritic cells is exposed to an antigen and allowed to process andpresent the antigen in vitro (this procedure is sometimes referred to inthe art as “antigen-pulsing”). An alternate method for preparingdendritic cells that present antigen is to transfect the dendritic cellswith a gene encoding an antigen-specific polypeptide. Once the dendriticcells express the antigen, the antigen-presenting dendritic cells can beadministered to a patient.

The invention also provides for the ex vivo preparation ofantigen-specific T cells. Following the procedures described above forpreparing large numbers of antigen-presenting dendritic cells ex vivo,the collected antigen-presenting dendritic cells are used to generateantigen-specific T cells from naive T cells that have been collectedfrom a patient. After the antigen has been adequately presented to the Tcells generated, the antigen-specific T cells can be administered to thepatient.

The invention also provides a method of augmenting an immune response ina patient that has an infectious disease wherein the method comprisesthe step of administering an amount of flt3-ligand sufficient toincrease the patient's number of dendritic cells. Embodiments of methodsfor augmenting an immune response include administering flt3-ligand incombination therapies with additional active compounds, including butnot limited to CD40 binding proteins, 4-1BB-L, antibodies to 4-1BB,interferon alpha, RANKL, a CD30 ligand antagonist, and combinationsthereof.

The invention also provides a method of augmenting an immune response ina patient that has a cancerous or neoplastic disease wherein the methodcomprises the step of administering an amount of flt3-ligand sufficientto increase the patient's number of dendritic cells. Embodiments ofmethods for augmenting an immune response include administeringflt3-ligand in combination therapies with additional active compounds,including but not limited to CD40 binding proteins, 4-1BB-L, antibodiesto 4-1BB, interferon alpha, RANKL, a CD30 ligand antagonist, andcombinations thereof. Such method provides a means to enhance thepatient's tumor-specific immune response.

A method for enhancing a patient's autoimmune tolerance wherein themethod comprises the step of administering an amount of flt3-ligandsufficient to increase the patient's number of dendritic cells. Furtherincluded are methods for promoting survival of grafts and transplantedtissues and organs.

The methods of the invention can further comprise the use of aneffective amount of a cytokine in sequential or concurrent combinationwith flt3-ligand. Such cytokines include, but are not limited to,interleukins (“ILs”) IL-3 and IL-4, a colony stimulating factor (“CSF”)selected from the group consisting of granulocyte macrophage colonystimulating factor (“GM-CSF”) or GM-CSF/IL-3 fusions, or other cytokinessuch as TNF-α, CD40 binding proteins (e.g. CD40-L), 4-1BB antagonists(e.g., antibodies immunoreactive with 4-1BB and 4-IBB-L), interferonalpha, RANKL, CD30 ligand antagonists (e.g., CD30 ligand monoclonalantibodies and soluble CD30-Fc fusion polypeptides), and c-kit ligand.

The invention further includes a dendritic cell expansion mediumcomprising cell growth medium, autologous serum, and flt3-ligand aloneor in combination with a cytokine from the group listed above.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to the use of flt3-ligand to generate largenumbers of intermediate cell types from CD34+ hematopoietic progenitorcells and stem cells. Such intermediate cell types include myeloidcells, monocytic cells, macrophages and dendritic cells. The largenumbers of these intermediate cell types are not naturally found in vivoand can be generated by administering flt3-ligand. Such enhancement inoverall cell number can augment the immune response to antigen in thehost. Another embodiment of the invention is the isolation and use ofsuch intermediate cell types as antigen-presenting cells or the use asvaccine adjuvants. The invention, while particularly focused on theembodiment concerning dendritic cells, is also applicable to myeloid,monocytic and macrophage cell types.

As used herein, the term “flt3-ligand” refers to a genus of polypeptidesthat are described in U.S. Pat. No. 5,554,512, EP 0627487 A2 and in WO94/28391, both incorporated herein by reference. A human flt3-ligandcDNA was deposited with the American Type Culture Collection, Rockville,Md., USA (ATCC) on Aug. 6, 1993 and assigned accession number ATCC69382. The deposit was made under the terms of the Budapest Treaty.Flt3-L can be made according to the methods described in the documentscited above.

As described in U.S. Pat. No. 5,554,512 at column 5, line 15, the term“flt3-L” encompasses proteins having the amino acid sequence 1 to 235 ofSEQ ID NO:2, as well as those proteins having a high degree ofsimilarity or a high degree of identity with the amino acid sequence 1to 235 of SEQ ID NO:2, and which proteins are biologically active andbind the flt3 receptor. In addition, the term refers to biologicallyactive gene products of the DNA of SEQ ID NO:1. Further encompassed bythe term “flt3-L” are the membrane-bound proteins (which include anintracellular region, a membrane region, and an extracellular region),and soluble or truncated proteins which comprise primarily theextracellular portion of the protein, retain biological activity and arecapable of being secreted. Specific examples of such soluble proteinsare those comprising the sequence of amino acids 28-160 of SEQ ID NO:2.

A “flt3-L variant” as referred to herein, means a polypeptidesubstantially homologous to native flt3-L, but which has an amino acidsequence different from that of native flt3-L (human, murine or othermammalian species) because of one or more deletions, insertions orsubstitutions. The variant amino acid sequence preferably is at least80% identical to a native flt3-L amino acid sequence, most preferably atleast 90% identical. The percent identity may be determined, forexample, by comparing sequence information using the GAP computerprogram, version 6.0 described by Devereux et al. (Nucl. Acids Res.12:387, 1984) and available from the University of Wisconsin GeneticsComputer Group (UWGCG). The GAP program utilizes the alignment method ofNeedleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smithand Waterman (Adv. Appl. Math 2:482, 1981). The preferred defaultparameters for the GAP program include: (1) a unary comparison matrix(containing a value of 1 for identities and 0 for non-identities) fornucleotides, and the weighted comparison matrix of Gribskov and Burgess,Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff,eds., Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for eachgap and an additional 0.10 penalty for each symbol in each gap; and (3)no penalty for end gaps. Variants may comprise conservativelysubstituted sequences, meaning that a given amino acid residue isreplaced by a residue having similar physiochemical characteristics.Examples of conservative substitutions include substitution of onealiphatic residue for another, such as Ile, Val, Leu, or Ala for oneanother, or substitutions of one polar residue for another, such asbetween Lys and Arg; Glu and Asp; or Gln and Asn. Other suchconservative substitutions, for example, substitutions of entire regionshaving similar hydrophobicity characteristics, are well known. Naturallyoccurring flt3-L variants are also encompassed by the invention.Examples of such variants are proteins that result from alternate mRNAsplicing events or from proteolytic cleavage of the flt3-L protein,wherein the flt3-L binding property is retained. Alternate splicing ofmRNA may yield a truncated but biologically active flt3-L protein, suchas a naturally occurring soluble form of the protein, for example.Variations attributable to proteolysis include, for example, differencesin the N- or C-termini upon expression in different types of host cells,due to proteolytic removal of one or more terminal amino acids from theflt3-L protein (generally from 1-5 terminal amino acids).

As described in U.S. Pat. No. 5,554,512 at column 8, line 7, an aspectof the invention is soluble flt3-L polypeptides. Soluble flt3-Lpolypeptides comprise all or part of the extracellular domain of anative flt3-L but lack the transmembrane region that would causeretention of the polypeptide on a cell membrane. Soluble flt3-Lpolypeptides advantageously comprise the native (or a heterologous)signal peptide when initially synthesized to promote secretion, but thesignal peptide is cleaved upon secretion of flt3-L from the cell.Soluble flt3-L polypeptides encompassed by the invention retain theability to bind the flt3 receptor. Indeed, soluble flt3-L may alsoinclude part of the transmembrane region or part of the cytoplasmicdomain or other sequences, provided that the soluble flt3-L protein canbe secreted.

Soluble flt3-L may be identified (and distinguished from its non-solublemembrane-bound counterparts) by separating intact cells which expressthe desired protein from the culture medium, e.g., by centrifugation,and assaying the medium (supernatant) for the presence of the desiredprotein. The presence of flt3-L in the medium indicates that the proteinwas secreted from the cells and thus is a soluble form of the desiredprotein.

Soluble forms of flt3-L possess many advantages over the native boundflt3-L protein. Purification of the proteins from recombinant host cellsis feasible, since the soluble proteins are secreted from the cells.Further, soluble proteins are generally more suitable for intravenousadministration.

Examples of soluble flt3-L polypeptides include those comprising asubstantial portion of the extracellular domain of a native flt3-Lprotein. Such soluble mammalian flt3-L proteins comprise amino acids 28through 182 of SEQ ID NO:2. In addition, truncated soluble flt3-Lproteins comprising less than the entire extracellular domain areincluded in the invention. Such truncated soluble proteins arerepresented by the sequence comprising amino acids 28-160 of SEQ IDNO:2. When initially expressed within a host cell, soluble flt3-L mayadditionally comprise one of the heterologous signal peptides describedbelow that is functional within the host cells employed. Alternatively,the protein may comprise the native signal peptide, such that themammalian flt3-L comprises 1 through 182 of SEQ ID NO:2. Isolated DNAsequences encoding soluble flt3-L proteins are encompassed by theinvention.

The term “IL-3” refers to a genus of interleukin-3 polypeptides asdescribed in U.S. Pat. No. 5,108,910, incorporated herein by reference.Such polypeptides include analogs that have amino acid sequences thatare substantially similar to the native human interleukin-3 amino acidsequences disclosed, for example, in EP publ. Nos. 275,598 and 282,185,each incorporated herein by reference. The term “IL-3” also includesanalogs and alleles of IL-3 molecules that exhibit at least some of thebiological activity in common with native human IL-3. Exemplary analogsof IL-3 are disclosed in EP Publ. No. 282,185. Other forms of IL-3include human IL-3[Pro⁸Asp¹⁵Asp⁷⁰], human IL-3[Ser⁸Asp¹⁵Asp⁷⁰] and humanIL-3[Ser⁸]. A DNA sequence encoding human IL-3 protein suitable for usein the invention is publicly available from the American Type CultureCollection (ATCC) under accession number ATCC 67747. The nomenclatureused herein with respect to amino acid sequences in brackets designateswhich amino acids differ from the native human form. For example, humanIL-3[Ser⁸Asp¹⁵Asp⁷⁰] refers to a human IL-3 protein in which amino acid8 has been changed to a serine residue, amino acid 15 has been changedto an aspartic acid residue and the amino acid 70 has been changed to anaspartic acid residue.

The term “IL-4” refers to a polypeptide as described in Mosley et al.,Cell 59:335 (1989), Idzerda et al., J. Exp. Med. 171:861 (1990) andGalizzi et al., Intl. Immunol. 2:669 (1990), each of which isincorporated herein by reference. Such IL-4 polypeptide includes analogsthat have an amino acid sequence that is substantially similar to thenative human IL-4 amino acid sequences described in Mosley et al.,Idzerda et al., and Galizzi et al. and which are biologically active inthat they are capable of binding to a IL-4 receptor, transducing abiological signal initiated by binding IL-4 receptor, or cross-reactingwith anti-IL-4 antibodies. The term “IL-4” also includes analogs ofnative human IL-4 molecules sufficient to retain biological activity ofnative human IL-4.

As used herein, “GM-CSF” refers to a genus of proteins as described inU.S. Pat. Nos. 5,108,910, and 5,229,496 each of which is incorporatedherein by reference. Such proteins include analogs that have an aminoacid sequence that is substantially similar to native human GM-CSF aminoacid sequences (e.g., as publicly available ATCC 53157 or ATCC 39900),and which are biologically active in that they are capable of binding toa GM-CSF receptor, transducing a biological signal initiated by bindingGM-CSF receptor, or cross-reacting with anti-GM-CSF antibodies. Aminoacid sequences are disclosed, for example in Anderson, et al., Proc.Natl. Acad. Sci., USA 82:6250 (1985). Commercially available GM-CSF(sargramostim, Leukine®) is obtainable from Immunex Corp., Seattle,Wash.). The term “GM-CSF” also includes analogs of the native humanGM-CSF molecules described in U.S. Pat. Nos. 5,108,910, and 5,229,496sufficient to retain biological activity of native human GM-CSF.Exemplary analogs of GM-CSF include, for example, those described in EPPubl. No. 212914 and WO 89/03881, each of which is incorporated hereinby reference. Other analogs of GM-CSF also may be used to constructfusion proteins with IL-3. A DNA sequence encoding a particularlypreferred GM-CSF protein having potential glycosylation sites removed ispublicly available from the ATCC under accession numbers ATCC 67231.

The term “GM-CSF/IL-3 fusion protein” means a C-terminal to N-terminalfusion of GM-CSF and IL-3. The fusion proteins are known and aredescribed in U.S. Pat. Nos. 5,199,942, 5,108,910 and 5,073,627, each ofwhich is incorporated herein by reference. A preferred fusion protein isPIXY321 as described in U.S. Pat. No. 5,199,942.

The term “c-kit ligand” also known as Mast Cell Growth Factor (MGF),Steel Factor or Stem Cell Factor (SCF), refers to a polypeptidedescribed in EP 423,980, which is incorporated herein by reference, andthat claims priority from U.S. patent application Ser. No. 589,701,filed Oct. 1, 1990. Such c-kit ligand polypeptide includes analogs thathave an amino acid sequence that is substantially similar to the nativehuman c-kit ligand amino acid sequences described in EP 423,980 andwhich are biologically active in that they are capable of binding to ac-kit receptor, transducing a biological signal initiated by bindingc-kit receptor, or cross-reacting with anti-c-kit ligand antibodies. Theterm “c-kit ligand” also includes analogs of native human c-kit ligandmolecules sufficient to retain biological activity of native human c-kitligand.

The term “CD40 binding protein” refers to polypeptides that bind CD40,including but not limited to CD40-L and antibodies immunoreactive withCD40, as described in PCT publications WO 93/08207 and WO 96/40918 eachof which is incorporated herein by reference. Such CD40 binding proteinsinclude analogs that have an amino acid sequence that is substantiallysimilar to the native human CD40-L amino acid sequences described in thePCT publications, and which are biologically active in that they arecapable of binding to CD40, transducing a biological signal initiated bybinding CD40, or cross-reacting with anti-CD40 antibodies. The term CD40binding proteins also includes analogs of native human CD40-L andantibodies reactive with CD40 that are sufficiently homologous to nativemolecules so as to retain biological activity of native CD40 bindingproteins.

The terms “4-1BB-L” and “antibody to 4-1BB” refer to molecules that aredescribed in U.S. Pat. No.5,674,704 and Alderson et al. Eur. J. Immunol.24:2219-2227, 1994, which are incorporated herein by reference. 4-1BB-Lincludes analogs that have an amino acid sequence that is substantiallysimilar to the native 4-1BB-L amino acid sequence described in the abovementioned publication and which are biologically active in that they arecapable of binding to 4-1BB or transducing a biological signal initiatedby binding 4-1BB, such as inducing a proliferative response instimulated primary T cells. The terms 4-1BB-L and antibodies to 4-1BBalso includes analogs of native 4-1BB-L and analogs of antibodiesreactive with 4-1BB that are sufficiently homologous to the nativecompounds so as to retain biological activity of 4-1BB-L and thedescribed antibodies.

The term “interferon alpha” refers to naturally occurring forms ofinterferon alpha (leukocyte interferon) as isolated from cells, celllines, tissue, and other sources, as well as recombinant forms,consensus forms, analogs, and variant forms that exhibit interferonalpha biological activity. Hybrid “interferon alpha A/D,” also called“Universal Type I Interferon,” has been described by Fisher et al.,Biochem Biophys. Res. Commun. 119(1):108 (1984), incorporated herein byreference. Interferon alpha polypeptides have been described in, interalia, Pestka et al., Ann. Rev. Biochem. 56:727 (1987), Goeddel et al.,Nature 290:20 (1980), Petska et al., in Human Cylokines, BlackwellScientific Publications 1-16 (1992), Petska, Semin. Oncol. 24(3):S9-4(197), U.S. Pat. No. 4,414,150, and WO 96/11953, all of which areincorporated herein by reference.

The term “CD30 ligand antagonist” refers to any molecule that interfereswith the binding between CD30 ligand and the CD30 receptor. CD30 ligandis described, inter alia, in U.S. Pat. No. 5,480,981 which isincorporated herein by reference. CD30 ligand antagonists include, butare not limited to, CD30 ligand antibodies (including CD30 ligandmonoclonal antibodies) and soluble CD30 polypeptides and analogs thereof(including soluble CD30-Fc fusion polypeptides). CD30 ligand antagonistsmay also be antisense nucleic acids, ribozymes, muteins, aptamers, andsmall molecules that interfere with the CD30-CD30 ligand interaction.

“RANK ligand” (“RANKL”) polypeptides are described in, inter alia, WO98/28426; “TRAIL” polypeptides are described in, inter alia, U.S. Pat.No. 5,763,223; “Tek” (also called Tie2, ork) polypeptides are describedin, inter alia, U.S. Pat. No. 5,447,860. These publications areincorporated herein by reference.

The term “adjuvant” refers to a substance that enhances, augments orpotentiates the host's immune response to a vaccine antigen.

The term “antagonist” includes various classes of molecules that arecapable of interfering with a specified biological interaction and/oractivity. Antagonists include, but are not limited to, antibodies,soluble forms of a target polypeptide, antisense nucleic acids,ribozymes, muteins, aptamers, and small molecules.

The procedure for “ex vivo expansion” of hematopoietic stem andprogenitor cells is described in U.S. Pat. No. 5,199,942, incorporatedherein by reference. Briefly, the term means a method comprising: (1)collecting CD34⁺ hematopoietic stem and progenitor cells from a patientfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo . In addition to the cellular growth factorsdescribed in U.S. Pat. No. 5,199,942, other factors such as flt3-ligand,IL-1, IL-3, c-kit ligand, can be used.

The term “immunogenicity” means relative effectiveness of an immunogenor antigen to induce an immune response.

The term “substantially similar” means a variant amino acid sequencepreferably that is at least 80% identical to a native amino acidsequence, most preferably at least 90% identical. Percent identity maybe determined by visual inspection. Percent identity may be determinedusing the alignment method of Needleman and Wunsch (J. Mol. Biol.48:443, 1970) as revised by Smith and Waterman (Adv. Appl. Math 2:482,1981). Preferably, percent identity is determined by using a computerprogram, for example, the GAP computer program version 10.x availablefrom the Genetics Computer Group (GCG; Madison, Wis., see also Devereuxet al., Nucl. Acids Res. 12:387, 1984). The preferred default parametersfor the GAP program include: (1) a comparison matrix containing a valueof 1 for identities and 0 for non-identities for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979 for amino acids; (2) a penalty of 30 (amino acids) or50 (nucleotides) for each gap and an additional 1 (amino acids) or 3(nucleotides) penalty for each symbol in each gap; (3) no penalty forend gaps; and (4) no maximum penalty for long gaps.

Variants may comprise conservatively substituted sequences, meaning thata given amino acid residue is replaced by a residue having similarphysiochemical characteristics. Examples of conservative substitutionsinclude substitution of one aliphatic residue for another, such as Ile,Val, Leu, or Ala for one another, or substitutions of one polar residuefor another, such as between Lys and Arg; Glu and Asp; or Gln and Asn.Other such conservative substitutions, for example, substitutions ofentire regions having similar hydrophobicity characteristics, are wellknown. Naturally occurring variants are also encompassed by theinvention. Examples of such variants are proteins that result fromalternate mRNA splicing events or from proteolytic cleavage of thenative protein, wherein the native biological property is retained.

As used herein, “vaccine” means an organism or material that contains anantigen in an innocuous form. The vaccine is designed to trigger animmunoprotective response. The vaccine may be recombinant ornon-recombinant. When inoculated into a non-immune host, the vaccinewill provoke active immunity to the organism or material, but will notcause disease. Vaccines may take the form, for example, of a toxoid,which is defined as a toxin that has been detoxified but that stillretains its major immunogenic determinants; or a killed organism, suchas typhoid, cholera and poliomyelitis; or attenuated organisms, that arethe live, but non-virulent, forms of pathogens, or it may be antigenencoded by such organism, or it may be a live tumor cell or an antigenpresent on a tumor cell.

A variety of cell selection techniques are known for identifying andseparating CD34⁺ hematopoietic stem or progenitor cells from apopulation of cells. Methods and materials for identifying and selectingsuch cell types are known. For example, monoclonal antibodies can beused to bind to a marker protein or surface antigen protein found onstem or progenitor cells. Such markers or cell surface antigens forhematopoietic stem cells include CD34 and Thy-1. In one method,antibodies are fixed to a surface, for example, glass beads, andcontacted with a mixture of cells suspected of containing stem cells.This permits the antibodies to bind and secure the stem cells to theglass beads. Alternatively, the antibodies can be incubated with thecell mixture and the resulting combination contacted with a surfacehaving an affinity for the antibody-cell complex. Undesired cells andcell matter are removed providing a relatively pure population of stemcells. Stem or progenitor cells having the CD34 marker constitute onlyabout 1% to 3% of the mononuclear cells in the bone marrow. The amountof CD34⁺ stem or progenitor cells in the peripheral blood isapproximately 10- to 100-fold less than in bone marrow.

With regard to the particular aspects of the invention, choosingsuitable stem or progenitor cell selection means will depend on thedesired phenotype of the cell to be isolated. Hematopoietic stem cellsare selectable by virtue of their physical characteristics, such asexpressing the membrane-bound flt3 receptor, or having the followingcellular markers: CD34 or Thy-1. Monoclonal antibodies that recognizeany of these antigens have been described in U.S. Pat. No. 4,714,680(anti-My-10) incorporated herein by reference, anti-CD34 is commerciallyavailable from Becton Dickinson, Franklin Lakes, N.J.), and anti-Thy-1monoclonal antibodies can be readily generated using the methodsdescribed by Dalchau et al., J. Exp. Med. 149:576 (1979), incorporatedherein by reference. A flt3 receptor binding protein also may be used,such as anti-flt3 monoclonal antibodies or the flt3-ligand. The cellbinding protein is brought into contact with the collected cell mixtureand the combination is allowed to incubate for a period of timesufficient to permit the binding of the desired cell to the cell bindingprotein.

An alternative means of selecting the quiescent stem cells is to inducecell death in the dividing, more lineage-committed, cell types using anantimetabolite such as 5-fluorouracil (5-FU) or an alkylating agent suchas 4-hydroxycyclophosphamide (4-HC). The non-quiescent cells arestimulated to proliferate and differentiate by the addition of growthfactors that have little or no effect on the stem cells, causing thenon-stem cells to proliferate and differentiate and making them morevulnerable to the cytotoxic effects of 5-FU or 4-HC. See Berardi et al.,Science, 267:104 (1995), which is incorporated herein by reference.

Isolation of the hematopoietic stem or progenitor cells can be performedby using, for example, affinity chromatography, antibody-coated magneticbeads, or antibodies fixed to a solid matrix, such as glass beads,flasks, etc. Antibodies that recognize a stem or progenitor cell surfacemarker can be fused or conjugated to other chemical moieties such asbiotin—which can be removed with an avidin or a streptavidin moietysecured to a solid support; fluorochromes useful in fluorescenceactivated cell sorting (FACS), or the like. Preferably, isolation isaccomplished by an immunoaffinity column. Immunoaffinity columns cantake any form, but usually comprise a packed bed reactor. The packed bedin these bioreactors is preferably made of a porous material having asubstantially uniform coating of a substrate. The porous material, whichprovides a high surface area-to-volume ratio, allows for the cellmixture to flow over a large contact area while not impeding the flow ofcells out of the bed. Typical substrates include avidin andstreptavidin, while other conventional substrates can be used. Thesubstrate should, either by its own properties, or by the addition of achemical moiety, display high-affinity for a moiety found on thecell-binding protein such as a monoclonal antibody. The monoclonalantibodies recognize a cell surface antigen on the cells to beseparated, and are typically further modified to present a biotinmoiety. It is well-known that biotin has a high affinity for avidin, andthe affinity of these substances thereby removably secures themonoclonal antibody to the surface of the packed bed. Such columns arewell known in the art, see Berenson, et al., J. Cell Biochem., 10D:239(1986). The column is washed with a PBS solution to remove unboundmaterial. Target cells can be released from the beads using conventionalmethods. Immunoaffinity columns of the type described above that utilizebiotinylated anti-CD34 monoclonal antibodies secured to an avidin-coatedpacked bed are described for example, in PCT Publ. No. WO 93/08268. Avariation of this method utilizes cell binding proteins, such as themonoclonal antibodies or flt3-ligand as described above,removably-secured to a fixed surface in the isolating means. The boundcell binding protein then is contacted with the collected cell mixtureand allowed to incubate for a period of time sufficient to permitisolation of the desired cells.

Alternatively, the monoclonal antibodies that recognize the cell surfaceantigens can be labeled with a fluorescent label, e.g., chromophore orfluorophore, and separated by cell sorting according to the presence ofabsence or the amount of labeled product.

The collected CD34⁺ cells are then exposed to either flt3-ligand aloneor flt3-ligand in concurrent or sequential combination with one or moreof the following cytokines: GM-CSF, TNF-α, IL-3, IL-4, c-kit-ligand,CD40-L, 4-IBB-L or GM-CSF/IL-3 fusion proteins. CD34⁺ cells then areallowed to differentiate and commit to cells of the dendritic lineage.The dendritic cells are collected and can either be (a) administered toa patient in order to augment the immune system and T-cell mediated orB-cell mediated immune responses to antigen, (b) exposed to an antigenprior to administration of the dendritic cells into a patient, (c)transfected with a gene encoding an antigen-specific polypeptide or (d)exposed to an antigen and then allowed to process and present theantigen, ex vivo, to T-cells collected from the patient followed byadministration of the antigen-specific T-cells to the patient.

More specifically, the invention provides for the use of an effectiveamount of flt3-ligand to increase or mobilize dendritic cells in vivo,for example, in the patient's peripheral blood or spleen. By increasingthe quantity of the patient's dendritic cells, such cells may themselvesbe used to present antigen to T cells. For example, the antigen may beone that already exists within the patient, such as a tumor antigen, ora bacterial or viral antigen. Flt3-L may be used, therefore, to boostthe patient's lymphocyte-mediated (e.g., T cell and B cell mediated) ormyeloid-mediated immune response to the already present antigens thuspotentially enabling a more effective antigen-presentation to thepatient's T cells.

Further, flt3-L may be used in combination therapies with one or moreadditional agents to enhance an immune response against tumor, viral orbacterial antigens. For example, CD40 binding proteins, which enhancethe ability of dendritic cells to process and present antigens toeffector T cells can be administered in combination with flt3-L todramatically enhance an immune response. Such immune responses caninclude responses against viral or bacterial antigens that areresponsible for infectious diseases and immune responses to tumorantigens. As described in Example 4, a surprising synergy between a CD40binding protein and flt3-L has been discovered for their combinedability to enhance anti-tumor responses. Represen-tative CD40 bindingproteins useful in combination therapy with flt3-L include CD40-L andantibodies immunoreactive with CD40 which are described in PCTpublications WO 93/08207 and WO 96/40918.

Additionally, 4-1BB-L and antibodies reactive with 4-1BB, both of whichare T-cell co-activation factors, can be administered in combinationwith flt3-L to dramatically enhance immune responses. 4-1BB-L andantibodies reactive with 4-1BB can be used in combination therapies toenhance immune responses to viral antigens and bacterial antigensresponsible for infectious diseases and to enhance immune responses totumor antigens. More particularly, as described in Example 5, when usedin a combination therapy there is a surprising synergy between flt3-Land a 4-1BB-L or antibodies to 4-1BB for anti-tumor immune responses.4-IBB-L and antibodies reactive with 4-1BB are described in U.S. Pat.No. 5,674,704. The surprising synergy in the above described combinationtherapies for their ability to dramatically enhance anti-tumor immuneresponses suggests that stimulating more than one mechanism or more thanone cell population is a promising approach to cancer treatment.

Additionally, interferon alpha, RANKL, or a CD30 ligand antagonist canbe administered in combination with flt3-L to dramatically enhanceimmune responses. As described in Example 6, when used in a combinationtherapy there is a surprising synergy between flt3-L and interferonalpha for anti-tumor immune responses.

Other molecules that may be used in combination with flt3-L according tothe present invention include IL-2, IL-12, IL-15, TRAIL, Fas ligand,VEGF antagonists, Tek antagonists, molecules that enhance dendritic cellfunction, survival, or expansion, molecules that enhance T cellactivation or differentiation, molecules that enhance dendritic cellmigration including various chemokines, molecules that increase theavailability of target cell antigens, such as apoptotic factors andmolecules that enhance MHC Class I presentation including the variousinterferons, angiogenesis inhibitors, inhibitors of immunosuppressivemolecules released by tumors including IL-10, VEGF, and TGF-β, andtumor-specific antibodies including toxin- or radio-labeled antibodies.

In addition to stimulating an immune response to an antigen that alreadyexists within the patient, flt3-ligand may be administered prior to,concurrently with or subsequent to administration of an antigen to apatient for immunization purposes. Thus, as a vaccine adjuvant,flt3-ligand can generate large quantities of dendritic cells in vivo tomore effectively present the antigen. The overall response is a strongerand improved immune response and more effective immunization to theantigen. Further, flt3-L may be administered as a vaccine adjuvant incombination with additional active compounds prior to, concurrently withor subsequent to administration of an antigen to a patient forimmunization purposes to enhance an immune response against tumor, viralor bacterial antigens. For example, CD40 binding proteins, such asCD40-L and antibodies to CD40 which enhance the ability of dendriticcells to present antigens to T cells can be administered in combinationwith flt3-L to dramatically enhance an immune response. Similarly,4-1BB-L, antibodies reactive with 4-1BB, interferon alpha, RANKL, orCD30 ligand antagonists can be administered in combination with flt3-Lto enhance an immune response and provide more effective immunization tothe antigen.

The systemic administration of flt3-ligand not only is effective as avaccine adjuvant, but as discussed supra., is effective in augmenting animmune response against previously existing antigens. For example, theinventors have shown that flt3-ligand administration to tumor-bearingmice results in at least a significant decrease in the growth rate ofthe tumor, and can result in tumor regression in a large proportion ofthe mice. The data are presented in more detail in Example 3. Flt3-Ltherefore is an important cytokine in the generation of an effectiveimmune response in vivo against antigen. Data in Examples 4-6demonstrate that when used in combination therapy with additionalagents, flt3-L can provide a dramatically enhanced immune response invivo against antigen.

Because of its ability to generate dendritic cells, flt3-ligand alsofinds use in promoting the survival of transplanted tissue or organs.When allogeneic organs or other tissue is transplanted into a host theycan transfer stem cells, immature dendritic cells, and mature dendriticcells from the donor. These cells are called passenger cells and suchcells can graft into the hematopoietic system of the host. Additionally,stem cells, immature dendritic cells, and mature dendritic cells fromthe host may graft to the donor organ or tissue.

It is possible then to establish a tolerance between the graft and thehost since the immature dendritic cells from the host and donor tissueinteract with T-cells from the “other side.” Such interaction mayinclude the deletion of T-cells that recognize the majorhistocompatability complex (MHC) molecules that the dendritic cellsexpress. In this way, the donor T cells are “screened” so that they failto recognize and react against the host (i.e., no graft versus hostdisease) and the host T-cells are screened so that they fail torecognize and react against the graft (i.e., no graft rejection). Thus,a mutual tolerance can be achieved, and the graft is accepted.Administration of flt3-ligand to the host or donor prior totransplantation would generate increased numbers of dendritic cells insuch host or donor and permit increased tolerance and survival of thegraft.

For the growth and culture of dendritic cells, a variety of growth andculture media can be used, and the composition of such media can bereadily determined by a person having ordinary skill in the art.Suitable growth media are solutions containing nutrients or metabolicadditives, and include those that are serum-depleted or serum-based.Representative examples of growth media are RPMI, TC 199, Iscovesmodified Dulbecco's medium (Iscove, et al., F. J. Exp. Med., 147:923(1978)), DMEM, Fischer's, alpha medium, NCTC, F-10, Leibovitz's L-15,MEM and McCoy's. Particular examples of nutrients that will be readilyapparent to the skilled artisan include, serum albumin, transferrin,lipids, cholesterol, a reducing agent such as 2-mercaptoethanol ormonothioglycerol, pyruvate, butyrate, and a glucocorticoid such ashydrocortisone 2-hemisuccinate. More particularly, the standard mediaincludes an energy source, vitamins or other cell-supporting organiccompounds, a buffer such as HEPES, Tris, that act to stabilize the pH ofthe media, various inorganic salts. Particular reference is made to PCTPubl. No. WO 95/00632, wherein a variety of serum-free cellular growthmedia is described, such disclosure is incorporated herein by reference.

For any of the ex vivo methods of the invention, peripheral bloodprogenitor cells (PBPC) and peripheral blood stem cells (PBSC) arecollected using apheresis procedures known in the art. See, for example,Bishop et al., Blood, vol. 83, No. 2, pp. 610-616 (1994). Briefly, PBPCand PBSC are collected using conventional devices, for example, aHaemonetics® Model V50 apheresis device (Haemonetics, Braintree, Mass.).Four-hour collections are performed typically no more than five timesweekly until, for example, approximately 6.5×10⁸ mononuclear cells(MNC)/kg patient are collected. The cells are suspended in standardmedia and then centrifuged to remove red blood cells and neutrophils.Cells located at the interface between the two phases (also known in theart as the buffy coat) are withdrawn and resuspended in HBSS. Thesuspended cells are predominantly mononuclear and a substantial portionof the cell mixture are early stem cells. The resulting stem cellsuspension then can be contacted with biotinylated anti-CD34 monoclonalantibodies or other cell-binding means. The contacting period ismaintained for a sufficient time to allow substantial interactionbetween the anti-CD34 monoclonal antibodies and the CD34 antigens on thestem cell surface. Typically, times of at least one hour are sufficient.The cell suspension then is brought into contact with the isolatingmeans provided in the kit. The isolating means can comprise a columnpacked with avidin-coated beads. Such columns are well known in the art,see Berenson, et al., J. Cell Biochem., 10D:239 (1986). The column iswashed with a PBS solution to remove unbound material. Target stem cellscan be released from the beads and from anti-CD34 monoclonal antibodyusing conventional methods. The stem cells obtained in this manner canbe frozen in a controlled rate freezer (e.g., Cryo-Med®, Mt. Clemens,Mich.), then stored in the vapor phase of liquid nitrogen. Ten percentdimethylsulfoxide can be used as a cryoprotectant. After all collectionsfrom the donor have been made, the stem cells are thawed and pooled.Aliquots containing stem cells, growth medium, such as McCoy's 5Amedium, 0.3% agar, and at least one of the expansion factors:recombinant human GM-CSF, IL-3, recombinant human flt3-ligand, andrecombinant human GM-CSF/IL-3 fusion molecules (PIXY321) atconcentrations of approximately 200 U/mL, are cultured and expanded at37° C. in 5% CO₂ in fully humidified air for 14 days. Optionally, humanIL-1α or IL-4 may be added to the cultures. The most preferredcombination of expansion factors comprises flt3-ligand plus either IL-3or a GM-CSF/IL-3 fusion protein.

For in vivo administration to humans, flt3-ligand can be formulatedaccording to known methods used to prepare pharmaceutically usefulcompositions. Flt3-L can be combined in admixture, either as the soleactive material or with other known active materials (e.g. CD40 bindingproteins, such as CD40-L or antibodies reactive with CD40, 4-1BB-L orantibodies reactive with 4-1BB, interferon alpha, RANKL, CD30 ligandantagonists), with pharmaceutically suitable diluents (e.g., Tris-HCl,acetate, phosphate), preservatives (e.g., Thimerosal, benzyl alcohol,parabens), emulsifiers, solubilizers, adjuvants and/or carriers.Suitable carriers and their formulations are described in Remington'sPharmaceutical Sciences, 16th ed. 1980, Mack Publishing Co. In addition,such compositions can contain flt3-ligand complexed with polyethyleneglycol (PEG), metal ions, or incorporated into polymeric compounds suchas polyacetic acid, polyglycolic acid, hydrogels, etc., or incorporatedinto liposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts or spheroblasts. Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance of flt3-ligand.

Flt3-L can be administered topically, parenterally, or by inhalation.The term “parenteral” includes subcutaneous injections, intravenous,intramuscular, intracisternal injection, or infusion techniques. Thesecompositions will typically contain an effective amount of theflt3-ligand, alone or in combination with an effective amount of anyother active material, e.g. those described above. Effective amounts, ordosages, and desired concentrations of flt3-L and active compounds (e.g.CD40-L and/or 4-1BB-L, antibodies reactive with 4-1BB, interferon alpha,RANKL, CD30 ligand antagonists) contained in the compositions may varydepending upon many factors, including the intended use, patient's bodyweight and age, and route of administration. Preliminary doses can bedetermined according to animal tests, and the scaling of dosages forhuman administration can be performed according to art-acceptedpractices. Keeping the above description in mind, typical dosages offlt3-ligand may range from about 10 μg per square meter to about 1000 μgper square meter. A preferred dose range is on the order of about 100 μgper square meter to about 300 μg per square meter.

In addition to the above, the following examples are provided toillustrate particular embodiments and not to limit the scope of theinvention.

EXAMPLE 1 Generation of Dendritic Cells

This Example describes a method for using flt3-ligand to generate largenumbers of dendritic cells ex vivo. Cells having the CD34⁺ phenotype areisolated as described above, for example, first by generating a buffycoat of cells using a procedure described supra. Cells from the buffycoat are then incubated with a CD34 specific monoclonal antibody. TheCD34⁺ cells which are selected then are cultured in McCoy's enhancedmedia with 20 ng/ml each of GM-CSF, IL-4, TNF-α, or 100 ng/mlflt3-ligand or c-kit ligand. The culture is continued for approximatelytwo weeks at 37° C. in 10% CO₂ in humid air. Cells then are sorted byflow cytometry for CD1a⁺ and HLA-DR⁺ expression. The combination ofGM-CSF, IL-4 and TNF-α, resulted in a six to seven-fold increase in thenumber of cells obtained after two weeks of culture. The combination offlt3-ligand and c-kit ligand resulted in an additive 12-13-fold increasein absolute cell numbers. This correlated with an 18-fold expansion witheither flt3-ligand or c-kit ligand or to a 34-fold expansion with thecombination of flt3-ligand and c-kit ligand. Phenotypic analysis of thecells showed that between 60-70% of the cells were HLA-DR⁺, CD86⁺, with40-50% of the cells expressing CD1 a in all factor combinationsexamined. The addition of flt3-ligand increased the absolute number ofCD1a⁺ cells by 5-fold. c-Kit ligand increased those cells by 6.7-foldand the combination of flt3-ligand and c-kit ligand by 11-fold.Functional analysis of the resultant cells in an MLR revealed that thepresence of flt3-ligand or c-kit ligand did not affect the stimulatorycapacity of the resultant dendritic cells while increasing the numbersattained.

EXAMPLE 2 Use of Flt3-L in Dendritic Cell Expansion

This Example describes a method for using flt3-ligand for dendritic cellexpansion. Prior to cell collection, it may be desirable to mobilize orincrease the numbers of circulating PBPC and PBSC. Mobilization canimprove PBPC and PBSC collection, and is achievable through theintravenous administration of flt3-ligand or sargramostim (Leukine®,Immunex Corporation, Seattle, Wash.) to the patients prior to collectionof such cells. Other growth factors such as CSF-1, GM-CSF, c-kit ligand,G-CSF, EPO, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, GM-CSF/IL-3 fusion proteins, LIF, FGFand combinations thereof, can be likewise administered in sequence, orin concurrent combination with flt3-ligand. Mobilized or non-mobilizedPBPC and PBSC are collected using apheresis procedures known in the art.See, for example, Bishop et al., Blood, vol. 83, No. 2, pp. 610-616(1994). Briefly, PBPC and PBSC are collected using conventional devices,for example, a Haemonetics® Model V50 apheresis device (Haemonetics,Braintree, Mass.). Four-hour collections are performed typically no morethan five times weekly until approximately 6.5×10⁸ mononuclear cells(MNC)/kg patient are collected. Aliquots of collected PBPC and PBSC areassayed for granulocyte-macrophage colony-forming unit (CFU-GM) contentby diluting approximately 1:6 with Hank's balanced salt solution withoutcalcium or magnesium (HBSS) and layering over lymphocyte separationmedium (Organon Teknika, Durham, N.C.). Following centrifugation, MNC atthe interface are collected, washed and resuspended in HBSS. Onemilliliter aliquots containing approximately 300,000 MNC, modifiedMcCoy's 5A medium, 0.3% agar, 200 U/mL recombinant human GM-CSF, 200u/mL recombinant human IL-3, and 200 u/mL recombinant human G-CSF arecultured at 37° C. in 5% CO₂ in fully humidified air for 14 days.Optionally, flt3-ligand or GM-CSF/IL-3 fusion molecules (PIXY 321) maybe added to the cultures. These cultures are stained with Wright'sstain, and CFU-GM colonies are scored using a dissecting microscope(Ward et al., Exp. Hematol., 16:358 (1988). Alternatively, CFU-GMcolonies can be assayed using the CD34/CD33 flow cytometry method ofSiena et al., Blood, Vol. 77, No. 2, pp 400-409 (1991), or any othermethod known in the art.

CFU-GM containing cultures are frozen in a controlled rate freezer(e.g., Cryo-Med®, Mt. Clemens, Mich.), then stored in the vapor phase ofliquid nitrogen. Ten percent dimethylsulfoxide can be used as acryoprotectant. After all collections from the patient have been made,CFU-GM containing cultures are thawed and pooled. The thawed cellcollection is contacted with flt3-ligand either alone, sequentially orin concurrent combination with other cytokines listed above. Suchexposure to flt3-ligand will drive the CFU-GM to dendritic cell lineage.The dendritic cells are reinfused intravenously to the patient.

EXAMPLE 3 Use of Flt3-L in Augmenting Anti-Tumor Immune Responses

This Example describes a method for using flt3-L to augment anti-tumorimmune responses in vivo. Female C57BL/10J (B10) mice (The JacksonLaboratory, Bar Harbor, Me.) were injected with 5×10⁵ viable B10.2fibrosarcoma tumor cells by intradermal injection in a midline ventralposition in a total volume of 50 μl. The fibrosarcoma B10.2 line is ofB10 origin and has been described previously, see Lynch et a., Euro. J.Immunol., 21:1403 (1991) incorporated herein by reference. Thefibrosarcoma B10.2 line was induced by subcutaneous implantation of aparaffin pellet containing 5 mg of methylcholanthrene. The tumor cellline was maintained in vitro in α-modified MEM containing 5% FBS, 2 nML-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin. Recombinanthuman flt3-L (10 μg/injection) was administered on a daily basis over a19-day period (unless otherwise noted) by subcutaneous injection in atotal volume of 100 μl. Control mice were similarly injected with asimilar volume of buffer containing 100 ng MSA. Tumor growth rates weredetermined by plotting the tumor size versus time after tumor challenge.Tumor size was calculated as the product of two perpendicular diameters,measured by calipers, and is expressed as the mean tumor size of onlythose mice bearing a tumor within a particular treatment group. Thenumber of mice bearing tumors compared to the number challenged for eachtreatment group at the termination of an experiment are shown in thedata below.

From Table I, the data is a compilation of six different experimentswherein B10.2 tumor-bearing mice were either treated with flt3-ligand orMSA. Complete tumor regression was observed in 19 of 50 flt3-ligandtreated mice compared to 1 of 30 in MSA-treated mice (p<0.0001 usingFishers Exact Test). The rate of tumor growth in flt3-ligand treatedmice (mean tumor size in tumor-bearing mice at week 5 post-tumorchallenge was 60+/−8 mm²) was significantly reduced (p.0001 by Analysisof Variance) compared to MSA-treated mice (mean tumor size at week 5post-tumor challenge was 185+/−17 mm²). TABLE I B10.2 Fibrosarcoma +/−Flt3-L Composite of Six Experiments Tumor Size (mm²) Weeks Post MSAControl Standard Flt3-L Standard Tumor Challenge (100 ng/day) Error (10μg/day) Error 0 0 0 0 0 1 25 2.6 24 2.2 2 62 7.5 49 3.6 3 98 10.6 49 3.94 149 14.5 50 5 5 185 16.8 60 8.4Tumor size was sharply retarded with flt3-L compared to the control.Therefore, the data show that flt3-L is an important cytokine in theaugmentation of the immune response against tumor and foreign antigens,and in particular against cancer.

EXAMPLE 4 Use of Flt3-L in Combination Therapy to Activate an ImmuneResponse

This Example demonstrates the use of flt3-L in combination with a CD40binding protein to augment anti-tumor immune responses in vivo. In onestudy C57BL/10J (B10) mice (The Jackson Laboratory, Bar Harbor, Me.)were injected intradermally with 5×10⁵ cells of the viable B10.2fibrosarcoma tumor cell line described in Example 3 above, and the micewere subdivided into four sets each containing eight mice. In one set ofmice, beginning on the same day as the tumor injections, recombinanthuman flt3-L (10 μg/injection/day) was administered to each mouse on adaily basis over a 20-day period by subcutaneous injection in a totalvolume of 100 μl. In another set of mice each mouse was injected withthe same volume and amount of CD40-L each day for 20 days. In a thirdset, each mouse was injected with a combination of 10 μg flt3-L and 10μg of CD40-L per day for 20 days. Control mice were similarly injectedwith a similar volume of buffer containing 100 ng MSA. Tumor growthrates were determined measuring tumor size each week after tumorchallenge over a 6 week period. Tumor size was calculated as the productof two perpendicular diameters, measured by calipers, and is expressedas the mean tumor size. Only mice bearing tumors within each group wereconsidered in determining the mean size. The frequency of tumorrejections was also determined and expressed as the number of micebearing no tumors compared to the number challenged for each treatmentgroup at the termination of an experiment.

Table II provides data in the form of mean tumor size in tumor bearinganimals, calculated once a week over a 6 week period post challenge.Table III details the percent frequency of tumor rejection for each setof mice over a 6 week period post challenge. The data demonstrate thatfor tumor bearing mice, the mean tumor size mice in mice treated withflt3-L and flt3-L in combination with CD40-L is comparable and less thanthe tumor size in tumor bearing control mice. Significantly, however,mice receiving the combination therapy experienced significantly higherfrequency of tumor rejection than mice receiving flt3-L or CD40-L alone.More specifically, 6 weeks post challenge, 62.5% of the mice receivingthe combination therapy experienced complete tumor rejection. Bycontrast, at 6 weeks post challenge, 25% of the mice receiving flt3-Lalone experienced complete tumor rejection and none of the micereceiving CD40-L alone or MSA experienced complete tumor rejection.TABLE II B10.2 Fibrosarcoma Tumor Size (mm²) Weeks Post CD40-L Tumor MSAControl Flt3-L (10 μg/ Flt3-L/CD40-L Challenge (100 ng/day) (10 μg/day)day) (each 10 μg/day) 1 28 24 25 23 2 68 51 62 46 3 135 64 107 59 4 23981 212 80 5 351 117 343 137 6 482 159 493 213

TABLE III B10.2 Fibrosarcoma % Frequency of Tumor Rejection Weeks PostCD40-L Tumor MSA Control Flt3-L (10 μg/ Flt3-L/CD40-L Challenge (100ng/day) (10 μg/day) day) (each 10 μg/day) 1 0 0 0 0 2 0 0 0 0 3 0 0 0 04 0 12.5 0 50 5 0 25 0 62.5 6 0 25 0 62.5

In another study C3H/HeN mice were injected intradermally with 5×10⁵cells of a very aggressive tumor, the 87 fibrosarcoma tumor cell line(generated by chronic exposure of C3H/HeN(MTV−) mice to ultravioletradiation). The mice were then subdivided into four sets, eachcontaining ten mice. In one set of mice, beginning the day after thetumor injections, recombinant human flt3-L (10 μg/injection/day) wasadministered to each mouse on a daily basis over a 20-day period bysubcutaneous injection. In another set of mice, each mouse was injectedwith the same volume and amount of CD40-L each day, beginning at day 7and continuing to day 20. In a third set, each mouse received acombination therapy of CD40-L and flt3-L. The combination therapyincluded 10 μg/day of flt3-L beginning the day after tumor injection andcontinuing until day 20 and 10 μg/day of CD40-L beginning at day 7 andcontinuing until day 20. Mice in a control group were similarly injectedwith a similar volume of buffer containing 100 ng MSA. Tumor growthrates were determined by measuring tumor size each week post tumorchallenge over a 6 week period. Tumor size was calculated as the productof two perpendicular diameters, measured by calipers, and is expressedas the mean tumor size. Only mice bearing tumors were considered indetermining the mean size. The frequency of tumor rejections was alsodetermine and expressed as the number of mice bearing no tumors comparedto the number challenged for each treatment group at the termination ofan experiment.

Table IV provides data in the form of mean tumor size in tumor bearinganimals, calculated once a week over a 6 week period post challenge.Table V details the percent frequency of tumor rejection for each set ofmice over a 6 week period post challenge. The data demonstrate that fortumor bearing mice, the mean tumor size mice in mice treated with flt3-Lin combination with CD40-L is significantly less than the tumor size intumor bearing control mice and mice bearing tumors in the groupsreceiving only flt3-L and only CD40-L. Significantly, mice receiving thecombination therapy experienced significantly higher frequency of tumorrejection than mice receiving flt3-L or CD40-L alone. More specifically,6 weeks post challenge, 50% of the mice receiving the combinationtherapy experienced complete tumor rejection. By contrast, at 6 weekspost challenge, 10% of the mice receiving flt3L alone experiencedcomplete tumor rejection and none of the mice receiving CD40-L alone orMSA experienced complete tumor rejection.

The observations described above demonstrate that a flt3-L and CD40-Lcombination therapy can dramatically up-regulate anti-tumor immuneresponses in vivo. The data indicate that a synergy exists betweenflt3-L and the CD40 binding protein, CD40-L, in that when used aloneflt3-L and CD40-L show little or no tumor rejection. In combination therejection is dramatic. In addition to synergy, studies indicated thatthe combination of CD40-L and flt3L indused expression of IL-12 mRNA inthe tumors. TABLE IV 87 Fibrosarcoma Tumor Size (mm²) Weeks Post MSATumor Control Flt3-L CD40-L Flt3-L/CD40-L Challenge (100 ng/day) (10μg/day) (10 μg/day) (each 10 μg/day) 1 23 23 30 29 2 54 53 49 34 3 10894 87 44 4 176 159 144 67 5 286 256 247 115 6 465 439 410 239

TABLE V 87 Fibrosarcoma % Frequency of Tumor Rejection Weeks Post MSATumor Control Flt3-L CD40-L Flt3-L/CD40-L Challenge (100 ng/day) (10μg/day) (10 μg/day) (each 10 μg/day) 1 0 0 0 0 2 0 0 0 0 3 0 0 0 30 4 010 0 40 5 0 10 0 50 6 0 10 0 50

EXAMPLE 5 Use of Flt3-L in Combination Therapy to Activate an ImmuneResponse

This Example demonstrates the use of flt3-L in combination with anantibody reactive with 4-1BB to augment anti-tumor immune responses invivo. In one study C57BL/10J (B10) mice (The Jackson Laboratory, BarHarbor, Me.) were injected intradermally with 5×10⁵ cells of the viableB10.2 fibrosarcoma tumor cell line described in Example 3 above. In oneset of mice, beginning on the same day as the tumor injections,recombinant human flt3-L (10 μg/injection/day) was administered to eachmouse on a daily basis over a 14-day period by subcutaneous injection ina total volume of 100 μl. In another set of mice each mouse was injectedIP with 100 μg of rat anti mu 4-1BB (clone m6) on days 3 and 6 posttumor challenge. In a third set, each mouse was injected with 100 μg ratanti mu 4-1BB clone m6 on days 13 and 16. A fourth set of mice wereinjected with a combination of 10 μg flt3-L on days 1-14 and 100 μg ofrat anti mu 4-1BB clone m6 on days 13 and 16 post tumor challenge.Control mice were injected with buffer containing 100 ng MSA. Tumorgrowth rates were determined measuring tumor size each week after tumorchallenge over a 5 week period. Tumor size was calculated as the productof two perpendicular diameters, measured by calipers, and is expressedas the mean tumor size in mm². Only mice bearing tumors within eachgroup were considered in determining the mean tumor size. The percentincidence of tumors was also determine and expressed as the number ofmice bearing tumors compared to the number challenged for each treatmentgroup at the termination of an experiment.

Table VI provides data in the form of mean tumor size in tumor bearinganimals, calculated once a week over an 8 week period post challenge.Table VII details the percent incidence of tumors for each set of miceover an 8 week period post challenge. The data demonstrate that fortumor bearing mice, the mean tumor size in mice treated with flt3-Lalone and the mean tumor size in mice treated with the anti 4-1BBregimen are similar. However, when flt3-L in combination with anantibody reactive with 4-1BB is administered to mice, mean tumor size intumor bearing mice is remarkably decreased. Specifically, at 5 weekspost tumor challenge, mice receiving the combination therapy had a meantumor size of 0, indicating 100% tumor rejection. This data is supportedby the numbers in Table VII which demonstrate that mice receiving thecombination therapy experienced significantly lower incidence of tumorsthan mice receiving flt3-L or 4-1BB antibody alone. More specifically,at 5 weeks post challenge, all of the mice receiving the combinationtherapy experienced complete tumor rejection (0% tumor incidence). Bycontrast, at 5 weeks post challenge, 70% of the mice receiving flt3-Lalone had tumors and 50% and 70% of the mice receiving 4-1BB antibodyalone had tumors. This data provides evidence that anti-4-1BB synergizeswith flt3-L in enhancing immune response. TABLE VI B10.2 FibrosarcomaTumor Size (mm²) anti 4-1BB Weeks Post MSA anti 4-1BB anti 4-1BB (days13 Tumor Control Flt3-L (100 μg, day (100 μg, days and 16) Challenge(100 ng/day) (10 μg/day) 3 and 6) 13 and 16) and flt3-L 1 28 15 25 25 202 60 35 55 60 35 3 85 35 55 40 15 4 125 45 60 50 8 5 200 55 70 40 0 6280 95 80 45 0 7 125 130 115 0 8 160 135 0

TABLE VII B10.2 Fibrosarcoma % Tumor Incidence Flt3-L and Weeks Post MSAanti 4-1BB anti 4-1BB anti 4-1BB Tumor Control Flt3-L (days 3 (days 13(days 13 Challenge (100 ng/day) (10 μg/day) and 6) and 16) and 16) 1 100100 100 100 100 2 100 100 100 100 90 3 100 100 90 100 80 4 100 90 80 5030 5 100 70 70 50 0 6 100 60 60 40 0 7 100 60 50 20 0 8 100 60 50 20 0

EXAMPLE 6 Use of Flt3-L in Combination Therapy to Activate an ImmuneResponse

This Example demonstrates the use of flt3-L in combination withinterferon alpha to augment anti-tumor immune responses in vivo. In onestudy the B10.2 fibrosarcoma tumor cell line (described in Example 3above) was implanted in C57BL/10J (B10) mice on day 0. One set of mice(n=10) was treated with recombinant human flt3-L (50 μg/day, bysubcutaneous injection) on days 10 to 29 post tumor challenge. Anotherset of mice (n=5) was treated with human interferon alpha (interferonalpha A/D; 60,000 U/day, by subcutaneous injection) on days 21 to 25post tumor challenge. A third set of mice (n=5) was treated with flt3-Lon days 10 to 29 and also with interferon alpha on days 21 to 25.Control mice were injected with buffer containing 100 ng MSA. Tumorgrowth rates were determined by measuring tumor size over a 7 weekperiod. Tumor size was calculated as the product of two perpendiculardiameters, measured by calipers, and is expressed as the mean tumor sizein mm². Only mice bearing tumors within each group were considered indetermining the mean tumor size. The percent incidence of tumors wasalso determined (i.e., the number of mice bearing tumors compared to thenumber challenged) for each treatment group.

Table VIII shows the mean tumor size in tumor bearing animals, and TableIX shows the percent incidence of tumors. This data demonstrates thatinterferon alpha synergizes with flt3-L in enhancing immune response inthe B10.2 tumor model. Most significantly, the tumor rejection rate was40% for flt3-L alone and 80% for the combination of flt3-L andinterferon alpha. TABLE VIII B10.2 Fibrosarcoma Tumor size (mm²) Flt3LIFNα MSA (50 μg/day, (60K U/day, FL + Days Post (100 ng/day) days 1-20)days 11-15) IFNα Tumor Challenge (n = 10) (n = 10) (n = 5) (n = 5) 7 1716 16 16 14 42 43 46 41 21 71 76 77 69 28 125 107 125 101 35 183 126 19890 42 238 131 267 55 49 322 219 364 186

TABLE IX B10.2 Fibrosarcoma % Tumor incidence Flt3L IFNα MSA (50 μg/day,(60K U/day, FL + Days Post (100 ng/day) days 1-20) days 11-15) IFNαTumor Challenge (n = 10) (n = 10) (n = 5) (n = 5) 7 100 100 100 100 14100 100 100 100 21 100 100 100 80 28 100 100 100 80 35 100 100 100 80 42100 80 100 60 49 100 60 100 20

1. A vaccine composition comprising: (a) as an adjuvant, Flt-3 ligand inan amount sufficient increase the number of dendritic cells in apatients in vivo; and (b) an antigen, wherein said Flt3-ligand comprisesan amino acid sequence that is at least 90% identical to amino acids28-Xaa of SEQ ID NO:2, wherein Xaa is an amino acid from 160-235 of SEQID NO:2, and wherein said Flt3-ligand binds Flt-3.
 2. The vaccinecomposition of claim 1, wherein said Flt3-ligand comprises amino acids28-Xaa of SEQ ID NO:2, wherein Xaa is an amino acid from 160-235 of SEQID NO:2.
 3. The vaccine composition of claim 1, wherein said Flt3-ligandcomprises amino acids 28-235 of SEQ ID NO:2.
 4. The vaccine compositionof claim 1, wherein said Flt3-ligand comprises amino acids 28-182 of SEQID NO:2.
 5. The vaccine composition of claim 1, wherein said antigen isselected from the group consisting of a tumor antigen, a bacterialantigen and a viral antigen.
 6. A method of immunizing a patient,comprising administering the vaccine composition of any of claim 1-5 toa patient in need thereof.
 7. The method of claim 6, wherein saidadministering is parenteral, topical or inhalation.